The PU-DMPA had a lower molecular weight than the PU-BDO . The melting enthalpy of the PU-DMPA was greater than that of the PU-BDO. Both the PUs exhibited elastomeric behaviors with a comparable elongation at break  that can be electrospun for use as a scaffold in soft tissue engineering. The PU was synthesized from polycaprolactone diol, hexamethylene diisocyanate, and dimethylolpropionic acid (DMPA) chain extender using two-step polymerization and designated as PU-DMPA. A control PU using 1,4-butanediol  as a chain extender was synthesized similarly and designated as PU-BDO. The chemical structure of the two PUs was verified by FT-IR and  Soft tissue loss caused by trauma, post-tumor resection, aging, etc., can affect aesthetics and even lead to exposure of vital structures of the body . The repThermoplastic polyesters such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), polycaprolactone (PCL) and their copolymers have been widely used for tissue engineering applications due to their hydrolytic degradability . The mecPolyurethanes are a class of polymers composed of soft and hard segments joined by urethane links . The sofScaffold materials may need certain functionality to meet their specific application requirements. For example, polymeric scaffolds to be used in contact with blood need to have proper blood compatibility, whereas hydrophilicity and antibacterial capability are important for those to be used as a wound dressing. Specific functionality can be provided if the polymer contains active functional groups such as carboxyl groups or amino groups. For example, Gong et al. prepared a polycarbonate-based polyurethane using dimethylolpropionic acid (DMPA), which contains a carboxyl group, as a chain extender. The carboxyl group-containing polyurethane was then modified by hyaluronan for better blood compatibility . NotablyPolyurethanes have been fabricated into porous scaffolds by a variety of techniques ,21,22. ATg). DMPA was used as a chain extender. Specifically, isocyanate-terminated PCL prepolymer was prepared in the first step and reacted with an equimolar amount of DMPA in the second step to form the polyurethane—that is, DMPA is the sole compound acting as the chain extender. A control polyurethane using 1,4-butanediol  as a chain extender was synthesized similarly under the same conditions. The chemical structure of the polyurethanes was confirmed by FTIR and 1H-NMR. Their mechanical tensile properties, hydrophilicity, and cytocompatibility were evaluated and compared. The polyurethane using DMPA as a chain extender was further electrospun to fabricate fibrous scaffolds. As a demonstration of the functionalizable carboxyl groups in the polyurethane, gelatin was incorporated to the scaffold by carbodiimide chemistry. Finally, we investigated the possibility of conjugation of hyaluronan on the surface of the functionalizable polyurethane.In this study, we aimed to develop a biodegradable, elastomeric, and functionalizable polyurethane for soft tissue engineering. PCL was selected as soft segments because of its hydrolyzability and low glass transition temperature (2), dimethyl sulfoxide-d6, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), gelatin from porcine skin, Ponceau S solution, and hyaluronan sodium salt (HA) were obtained from Sigma-Aldrich . Dibutylamine was obtained from J.T. Baker . 1,1,1,3,3,3-Hexafluoro-2-propanol (99%) was obtained from Matrix Scientific . 2,2-Bis(hydroxymethyl)propionic acid (DMPA), 1,4-diaminobutane  and N-hydroxysuccinimide (NHS) were obtained from Alfa Aesar . Tetrahydrofuran (THF) was purchased from Macron . Poly-L-lysine  was kindly provided by Prof. Jeng-Shiung Jan. PCL diol was dried at 120 °C under vacuum for 4 h prior to use. 1,4-BDO and HDI were purified by distillation and stored over molecular sieves . DMAc was dried over molecular sieves for more than two days. The water content of PCL diol, HDI, 1,4-BDO, and DMAc was determined by a Karl Fischer titrator  to ensure that the water content is less than 100 ppm. DMPA and all other reagents were used as received. For cell culture, Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin, and trypsin-EDTA were obtained from Thermo Fisher Scientific  and fetal bovine serum was purchased from Hyclone .Polycaprolactone diol  was obtained from Daicel (Japan). Hexamethylene diisocyanate  was obtained from Fluka (Switzerland). 1,4-Butanediol  was obtained from Acros . Dimethyl acetamide  was obtained from Tedia . Stannous octoate (Sn(Oct)w/v) solution of PCL diol in DMAc was heated to 65 °C in the reactor with continuous stirring. HDI and a catalytic amount of Sn(Oct)2 were then added. The prepolymerization reaction was allowed to continue at 65 °C until the isocyanate content reached the theoretical value determined by dibutylamine titration. Subsequently, in the second step, a solution of DMPA or 1,4-BDO was added to the prepolymer solution, initiating chain extension. The reaction was continued until the NCO peak at 2270 cm−1 in the Fourier transform infrared (FT-IR) spectrum of the reaction mixture was not detected. The product was precipitated in distilled water, filtered, and dried under vacuum at 60 °C for 24 h. The polymer was designated as PU-DMPA or PU-BDO according to the chain extender used.In this study, polyurethanes were synthesized by two-step solution polymerization . The synw/v) solutions. The solution was then poured into a Teflon dish (7.5 cm in diameter) and degassed under vacuum at room temperature. Upon the removal of bubbles, the PU-DMPA and the PU-BDO solutions were dried in a fume hood at room temperature and under vacuum at 60 °C, respectively, for 24 h, to form films. The thickness of the film was measured by a custom high-frequency ultrasound system.The PU-DMPA and the PU-BDO were dissolved in HFIP and DMF, respectively, to prepare 5% (The molecular weights of the PU-DMPA and the PU-BDO were determined by a gel permeation chromatography and light-scattering (GPC–LS) system  equipped with two Shodex HFIP columns and three detectors including RI , right angle light scattering, and viscometer . HFIP at a flow rate of 1.0 mL/min was used as the eluent.−1 to 4000 cm−1 at the resolution of 2 cm−1 using a Nicolet 6700 FT-IR spectrometer  with an ATR module.The chemical structure of the PU-DMPA and the PU-BDO was examined by infrared spectroscopy. FT-IR spectra of samples were obtained by scanning the samples for 64 times in the wave number range from 600 cm6 to prepare 20 mg/mL solutions. 1H NMR spectra (500 MHz) were recorded on an Avance 500 NMR spectrometer . Chemical shifts were given in ppm with tetramethylsilane as the standard.Samples of the PU-DMPA and the PU-BDO were dissolved in dimethyl sulfoxide-dThe melting behavior and crystallization of the PU-DMPA and the PU-BDO were investigated by a Perkin-Elmer DSC7 . Samples weighted 6 mg were examined. The sample chamber was purged with nitrogen gas at a flow rate of 50 mL/h. The temperature sequence was arranged as follows: the sample was heated to 280 °C at 10 °C/min, equilibrated at 280 °C, and cooled to −40 °C at 10 °C/min. Then the sample was heated again to 280 °C at 10 °C/min.The mechanical behavior of the PU-DMPA and the PU-BDO was estimated by uniaxial mechanical tensile testing. Dog-bone shaped specimens were punched from the PU-DMPA and the PU-BDO cast films using a miniature ASTM D412-C die . The specimen was mounted on an ATS machine  equipped with a 200 N load cell . A tensile test was conducted on under a crosshead speed of 30 mm/min until membrane break. The force (N) and displacement (mm) were recorded. The engineering stress was calculated as force divided by original cross-sectional area and the stretch ratio was calculated as (displacement + 16.5)/16.5. The stress–stretch curves of the specimen were plotted and the ultimate stress, elongation at break and initial modulus of the specimen were obtained.The wettability of the PU-DMPA and the PU-BDO cast films was assessed by water contact angle measurements. Water drops of 8 µL were generated by a micropipette and carefully deposited on the surface of the specimens. Photographs of the droplets on the surface were acquired by a digital camera and the contact angles were measured by ImageJ (NIH) with an average of six measurements for each sample.2 incubator. Before reaching confluence, cells were detached by 0.05% trypsin-EDTA, counted, and resuspended in culture medium for experiments. The PU-DMPA and the PU-BDO cast films were sterilized by immersion in 70% ethanol for 30 min, followed by washing with PBS and then exposure to UV irradiation in a laminar flow hood overnight. The HUASMCs were then seeded on the top of the film or in tissue culture polystyrene dishes at a density of 2.5 × 104/cm2. After 24 h, the cell seeded film was washed with PBS and then fixed by 4% paraformaldehyde for 30 min. Images of the attached cells on the film were acquired with an inverted optical microscope  equipped with a digital camera .Human umbilical artery smooth muscle cells (HUASMCs) were used to investigate cell adhesion behavior on the surface of the PU-DMPA and the PU-BDO cast films. HUASMCs were isolated according to an established protocol  and cultw/v) gelatin in PBS solution (10 mL) to allow the activated carboxyl groups to react with the amino groups in gelatin for 4 h at 40 °C  was placed in a solution of EDC (0.2 g) and NHS (0.05 g) in MES buffer  (10 mL) for 30 min to activate carboxyl groups on the film surface. After a brief water in MES buffer, the film was transferred to a 10%  solution. The solution was delivered by the syringe pump at a flow rate of 1.5 mL/h to the spinneret, on which a positive charge of ~10 kV was applied, and electrospun onto the rotating drum. A 25 cm tip-to-drum distance was employed. Fibers were collected for 2 h to form a membrane. The thickness of the membranes, measured by the high-frequency ultrasound system, ranged between 170 and 220 μm. Immobilization of gelatin on the electrospun PU-DMPA membrane was performed following the procedure described earlier. The result was also verified by FT-IR and Ponceau S staining. The porosity of the PU-DMPA electrospun membranes, before and after gelatin immobilization, was measured by a water displacement method [PU-DMPA fibrous membranes were prepared by electrospinning using a custom setup which consists of a spinneret (21-gage needle), a syringe pump , a high-voltage DC power supply , and a grounded aluminum rotating drum (102 mm in diameter) driven by a high-speed DC motor . Brieflyt method . Briefly−8 Pa. The diameters of electrospun PU-DMPA fibers, before and after gelatin immobilization, were measured using ImageJ with an average of 30 measurements from three SEM images of each polyurethane.The morphology of electrospun PU-DMPA fibers before and after gelatin immobilization was examined by scanning electron microscopy (SEM). Pieces of the polyurethane membranes (3 mm × 5 mm) were mounted onto stubs and sputter-coated with platinum . SEM images were taken by a field emission scanning electron microscope . The voltage used was 15 kV. The vacuum was 5 × 10w/v) solution. An excess amount of EDC (0.72 g) and NHS (0.18 g) in methanol (12 mL) was gradually added to the PU-DMPA solution. After reaction for 30 min, a solution of poly-L-lysine (0.06 g) in methanol (1.5 mL) was added to the solution. The reaction continued for 24 h with agitation. The solution was then casted in a Teflon dish and dried at room temperature to obtain a membrane, designated as PU-DMPA-PLL. The result of amino functionalization was verified by FT-IR and Ponceau S staining.Based on w/v) HA in MES buffer; the final concentrations of EDC and NHS were 6% (w/v) and 1.5% (w/v), respectively. The amino-functionalized PU-DMPA film , either PU-DMPA-BDA or PU-DMPA-PLL, were added to the HA buffer (10 mL) with stirring at 40 °C for 24 h.For immobilization of hyaluronic acid (HA) on the amino-functionalized PU-DMPA, EDC and NHS were added to 0.5% (v/v)) was centrifuged  at 1500 rpm for 15 min to obtain plasma. The PU-DMPA, PU-DMPA-BDA, PU-DMPA-PLL, PU-DMPA-BDA-HA and PU-DMPA-PLL-HA membranes with a size of 1 cm × 1 cm were incubated in the plasma at 37 °C for 1 h. The membranes were then washed with PBS to remove unattached platelets and fixed with a 2.5% glutaraldehyde solution at 4 °C for 2 h. Upon drying through graded alcohols, the membranes were sputter-coated with platinum and the morphology of platelet on the surfaces of the membranes was examined by scanning electron microscopy.Human whole blood was collected from healthy human donors. Anticoagulated blood  ester or urethane carbonyl groups, respectively [−1 was strong and broad compared to the two peaks for the PU-BDO. Note that, in addition to the carboxyl group in soft segments and urethane groups in hard segments, there are carboxyl groups in the hard segments and all these groups might contribute to H-bonded carbonyl groups. Both the PU-BDO and the PU-DMPA exhibited 2936 cm−1 and 2861 cm−1 absorptions, corresponding to symmetric and asymmetric C-H stretching from CH2, respectively. Generally, C=O stretching vibration of esters and urethanes manifests itself as peaks between 1600 cmectively ,31. In t−1 were assigned to N-H stretching of urethane amide (for both the PU-DMPA and the PU-BDO) or O-H stretching in the carboxyl group (for the PU-DMPA only). The shifting of the peaks to the low wave number for both the PU-DMPA and the PU-BDO also indicates the formation of hydrogen bonding.The peaks around 3300 cm1H-NMR spectra of the PU-DMPA and the PU-BDO with each peak assigned. The PU-DMPA and the PU-BDO spectra shared many common peaks, including: 1.2 ppm (peak a), 1.3 ppm (peak b), 1.5 ppm (peaks c-e), 2.25 ppm (peak f), 2.95 ppm (peak g), 3.6 ppm (peak h), 3.98 ppm (peak i), and 4.12 ppm (peak j). These peaks were assigned to proton signals of HDI and PCL units. Peaks d’ and j’ in the PU-BDO spectrum were assigned to proton signals of 1,4-BDO units, whereas peaks a’ and i’ in the PU-DMPA spectrum were DMPA, indicating the chain extenders were incorporated into the polymers, respectively. The results of 1H-NMR were consistent with the findings in the FT-IR; both support that both the polyurethanes were synthesized as designed.Tg) of both the polyurethanes were below room temperature, which indicates that both the polyurethanes are in a rubbery state at room temperature or under physiological conditions. The PU-DMPA had a slightly lower Tg (2.5 °C) compared to the PU-BDO (6.8 °C). Both the PU-DMPA and the PU-BDO displayed melting peaks, which is due to the crystallinity of PCL segments. Similarly, the melting temperature (Tm) of the PU-DMPA (39.2 °C) was slightly lower than that of the PU-BDO (41.3 °C). The reduction in Tg and Tm might be attributed to the compromised crystallinity of PCL soft segments caused by hydrogen bonds between the hard segments  and the soft segments (PCL carbonyl groups) [ groups) . Additiog et al. . The elag et al. . The greg et al. . The incg et al.  and bloog et al. , and it Scaffold materials for tissue engineering should have proper surface hydrophilicity for cells to adhere to. A moderate surface wettability (50–70°) has been shown to favor cell adhesion and growth . Figure After gelatin modification, the water contact angle of the PU-DMPA films slightly decreased to 57 ± 2.8°. The reduction in contact angle was not significant, however. w/v). The presence of carboxyl groups in the PU-DMPA was demonstrated by immobilization of gelatin on the as-spun PU-DMPA fibrous membrane using carbodiimide chemistry. The gelatin treatment significantly decreased the porosity of as-spun membranes from 78 ± 3% to 67 ± 7%. p < 0.05).We attempt to use the PU-DMPA for fabricating tissue engineering scaffolds. As electrospinning has many advantages over conventional methods in this regard , efforts were made to fabricate electrospun PU-DMPA fibrous membranes. Despite the relatively low molecular weight of the PU-DMPA, bead free and drop free electrospun fibers were obtained when the concentration of the PU-DMPA solution was increased to 15%  and the PU-DMPA-PLL-HA (28.6 ± 3.7°) cast films decreased significantly compared to that of the PU-DMPA-BDA (54.9 ± 4.0°) and PU-DMPA-PLL (54.0 ± 2.2°) films, respectively. For more functionalization routes of the PU-DMPA, we endowed the PU-DMPA with amino groups using carbodiimide chemistry. The amino functionalization was performed on the PU-DMPA film surface or in a homogeneous solution of the PU-DMPA in THF. The HA-modified surface of the PU-DMPA-BDA and the PU-DMPA-PLL films were subjected to platelet adhesion tests with the PU-DMPA, PU-DMPA-BDA, and PU-DMPA-PLL films serving as controls. A material that has antithrombotic ability might be applied in the development of blood contact devices. Caution must be taken as surface modification allows imparting materials with specific functions, but the effect fades out as the material surface degrades. Incorporation of pendant reactive groups by bulk modification for subsequent functionalization would be more endurable. In this study, both approaches were performed to amino-functionalize the PU-DMPA. The immobilization of HA on the amino-functionalized PU-DMPA film was performed by surface modification, however.2 may be replaced by amine catalysts such as triethylenediamine, which can be removed by water. Finally, due to the improved hydrophilicity of the PU-DMPA compared with the PU-BDO, the degradation rate of the PU-DMPA might be increased. The biodegradability of the PU-DMPA compared to the PU-BDO may be worth a thorough investigation. There are a few limitations in this study. The number of carboxyl groups in the PU-DMPA was determined by the DMPA content. In this study, isocyanate-terminated PCL prepolymer was formed prior to the reaction with equimolar amount of DMPA, which resulted in an alternating copolymer—that is, the carboxyl groups were evenly distributed in the PU-DMPA backbone. We did not attempt to manipulate the DMPA content by mixing PCL diol with DMPA when forming the prepolymer nor by changing the ratio of PCL diol and DMPA chain extender, which could result in block copolymers with unpredictable structures. The changes in the DMPA content might increase the functionalizability of the polyurethane but could alter the mechanical properties of the polyurethane, which deserves a systematic investigation. It was suggested an increase in the DMPA content could reduce the molecular weight of the polymer , howeverThe PU-DMPA, based on a PCL soft segment, a HDI hard segment and a DMPA chain extender, was synthesized successfully as designed. The time required to complete the chain extension of the PU-DMPA was tremendously longer than that of the control PU-BDO (4 days vs. 24 h) and the PU-DMPA had a much lower molecular weight than the PU-BDO. The melting enthalpy of the PU-DMPA was greater than that of the PU-BDO. The PU-DMPA had a higher initial modulus and a lower linear modulus and ultimate strength than the PU-BDO, whereas the elongation at break of both the polyurethanes were comparable. The PU-DMPA was more hydrophilic than the PU-BDO. The different properties and behaviors might be attributed to the carboxyl groups in the PU-DMPA. Both the polyurethanes exhibited no cytotoxicity. The improved hydrophilicity of the PU-DMPA manifested better cell adhesion. Bead free and drop free electrospun PU-DMPA fibrous membranes with a narrow fiber diameter distribution were successfully fabricated using a relatively concentrated PU-DMPA solution.Because of the presence of carboxyl groups in the PU-DMPA backbone, compounds containing amino groups can be conjugated to the PU-DMPA using carbodiimide chemistry. Compounds containing carboxyl groups, on the other hand, can be immobilized to the amino-functionalized PU-DMPA. Gelatin was successfully conjugated to the electrospun PU-DMPA membrane using carbodiimide chemistry. HA was immobilized on the amino-functionalized PU-DMPA cast film. Reduced platelet adhesion of the HA-modified PU-DMPA was noted. The PU-DMPA has the potential to be used as a scaffold material for soft tissue engineering.


Background. This study evaluated the incorporation of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), calcium sodium phosphosilicate bioactive glass (BAG), chitosan (CH), and methacryloyloxydodecylpyridinium bromide (MDPB) on the compressive and flexural strength, fluoride (F‒ ) release, and bacterial adhesion of conventional glass-ionomer cement (C-GIC). Methods. Modifications were implemented by adding CPP-ACP, BAG, and CH to the glass powder, while MDPB-GIC was prepared by incorporating MDPB to the liquid of C-GIC. Custom-made molds were used for specimen preparation. Compressive and flexural strengths were evaluated using a universal testing machine. F‒ release was calculated with Erichrome cyanide reagent, using UV-spectrophotometry, at two time intervals of 24 hours and seven days. For bacterial adhesion, the test specimens were exposed to the bacterial suspension of Streptococcus mutans and Lactobacillus acidophilus for 4 hours, and the adherent bacteria were quantified using colorimetry as the optical density (OD). Results. The incorporation of MDPB increased the flexural strength of C-GIC, with no effect on its compressive strength. CH significantly improved the compressive and flexural strength; modifications with CPP-ACP, BAG, and MDPB significantly improved the flexural strength of C-GIC. While MDPB-GIC released significantly higher F‒ at 24 hours, CPP-ACP- and BAG-modified GICs were comparable to C-GIC on day 7. C-GIC exhibited the highest bacterial adhesion, and MDPB-GIC showed the least. The data were analyzed with one-way (ANOVA), and pairwise comparisons were made with Tukey HSD tests. Conclusion. Hence, it can be concluded that the incorporation of CPP-ACP, BAG, and CH improved the mechanical properties of C-GIC, whereas MDPB improved the resistance of C-GIC to bacterial adhesion. Despite these advantages, GIC has certain drawbacks, such as brittleness and porosity, which result in poor mechanical properties, such as low wear resistance and fracture toughness.2Recurrent caries has been the most frequent cause of failure of dental restorations. Fluoride-releasing restorative materials were introduced to overcome this disadvantage. Among the various commercially available fluoride-releasing materials, glass-ionomer cement (GIC) has the highest fluoride release. Resin-modified glass-ionomer cement (RMGIC), compomer, and alkasite, or ion-releasing composite have evolved over the years to harness the advantages of composite resins and GIC. However, the fluoride release of these newer materials is still less than GIC.‒) ions present in GIC, which produces a stabilized amorphous calcium fluoride phosphate phase.3The composition of GIC has been experimented with the incorporation of a wide variety of biologically active materials. Modifications of GIC with casein phosphopeptide-amorphous calcium phosphate nanocomplexes (CPP-ACP), calcium sodium phosphosilicate bioactive glass (BAG), and chitosan (CH) have been reported in the literature. The incorporation of CPP-ACP to GIC has shown that the localization of CPP to amorphous calcium phosphate of the tooth surface increases the anticariogenicity by maintaining a state of supersaturation concerning the tooth mineral. Its anticariogenic effect is further potentiated by its interaction with fluoride  considerably improved its flexural strength and F7 Imazato et al confirmed the bactericidal activity of MDPB-modified monomer against seven species of oral streptococci.8Methacryloyloxydodecylpyridinium bromide (MDPB) is a quaternary ammonium compound obtained by substituting the hydroxyl group at the terminal end of hydroxy dodecylpyridinium bromide with the methacryloyl group. This increases the antibacterial activity of the parent compound. MDPB has been a breakthrough in the development of non-agent-releasing antibacterial restoratives.‒ release from GIC further deteriorates its surface integrity, favoring the adhesion of oral flora.9 Several studies have shown that incorporating MDPB into resinous materials like bonding agents and composite resins, resulted in a significant decrease in recurrent caries and bacterial adhesion to the biomaterial surface.10 No study has evaluated the effect of incorporating MDPB on the mechanical properties, F‒ release, and bacterial adhesion of C-GIC. Hence, this in vitro study aimed to evaluate the effect of incorporating CPP-ACP, BAG, CH, and MDPB on the compressive and flexural strength, F‒ release, and bacterial (Streptococcus mutans and Lactobacillus acidophilus) adhesion of C-GIC.Despite improvements in the mechanical properties of various modified formulations of GIC, bacterial adhesion on its surface still remains a concern as it predisposes the microenvironment at the tooth‒restoration interface to secondary caries. FThe chemicals used to prepare MDPB were of analytical grade. 1-bromododecane and 3-hydroxypyridine were procured from Spectrochem Pvt. Ltd., Mumbai, India. Hydroquinone, acrylic acid, and benzoyl chloride were procured from Merck, Mumbai, India. MDPB was synthesized by the reaction of hydroxy dodecylpyridinium bromide and methacryloyl chloride.0.72 g of acrylic acid was dissolved in 10 mL of distilled ethanol. This solution was added to 1.4 g of benzoyl chloride under 5 mL of 0.1 g of hydroquinone at a temperature of 72‒76°C. The obtained product, acryloyl chloride, was again dissolved in methanol and further subjected to re-distillation at a temperature of 72‒76°C to obtain methacryloyl chloride. Hydroquinone was added as a catalyst to prevent the polymerization of acrylic acid. Hydroxy dodecylpyridinium bromide was prepared from 1-bromododecane and 3-hydroxypyridine by the reflux method. Finally, MDPB was prepared by mixing the prepared hydroxy dodecylpyridinium bromide and methacryloyl chloride at a ratio of 1:1. The obtained product, MDPB, was further confirmed by an infrared (IR) spectrometer. The obtained IR spectrum of the prepared compound was compared with the standard compound, and the frequencies were assigned .C-GIC (group I) was prepared by dispensing two scoops of powder and two drops of liquid  on a moisture-impervious paper pad and mixing with an agate spatula in a folding motion for 25‒30 seconds to obtain a paste-like consistency.TM, GC Corporation, Japan), BAG powder , and 36 mg of CH powder  to 180 mg of type II GIC powder on separate paper pads and mixing the ingredients thoroughly. To these powders, 120 mg of type II GIC liquid was added and mixed using an agate spatula in a folding motion for 25‒30 seconds to obtain a paste-like consistency. MDPB-GIC (group V) was prepared by first mixing 60 mg of MDPB liquid and type II GIC liquid thoroughly. To this, 360 mg of type II GIC powder was then incorporated and mixed to obtain a paste-like consistency.Modifications of GIC with CPP-ACP (group II), BAG (group III), and CH (group IV) were implemented by dispensing 180 mg of CPP-ACP paste  were prepared for compressive strength and flexural strength testing, respectively. Fifteen specimens per group were prepared from the experimental materials for each strength test. During setting, the top and bottom portions of the molds were covered with Mylar strips to obtain a smooth surface. After the initial setting, the test specimens were removed from the molds and stored in artificial saliva for 24 hours. They were then subjected to compressive and flexural loading in a universal testing machine  at a 1 mm/min crosshead speed.‒ release was evaluated without replenishing the water, at two time intervals of 24 h and seven days with UV spectrophotometry  using Erichrome cyanide  as a reagent.Under ISO specification 19448:2018, five specimens measuring 6 × 4 mm were prepared using stainless steel molds for each group. After the initial setting, the specimens were stored in 100 mL of deionized distilled water at 37°C. FS. mutansand L. acidophilus, the common cariogenic oral bacteria, were used. After 4 hours, the test materials were retrieved from the culture broth and washed three times with 5 mL of sterile saline solution to remove non-adhering cells. The test materials were then suspended in glass tubes containing 1 mL of saline solution, and the tubes were transferred to an ultrasonic bath cleaner fitted with a test tube holder, operating at 47 kHz (234 W) and sonicated for 6 minutes to detach the adherent bacteria from the biomaterial surfaces, bringing them into suspension. The test specimens were then removed, and 10 mL of fresh broth was added to each tube. The tubes were again incubated at 37°C for 24 hours. After incubation, the concentration of the bacteria in the broth was finally measured using colorimetry . The results were tabulated as optical density (OD) values.For the bacterial adhesion tests, apart from the experimental groups, the polystyrene strip served as a positive control (group VI), and plastic mesh served as a negative control (group VII). Twenty specimens measuring 6 × 4 mm were prepared in each group and randomly divided into two subgroups (n = 10) each, based on the two bacterial strains tested. After the initial setting, the cement specimens were stored in artificial saliva for 24 hours. They were then sterilized in an autoclave at 121°C for 15 minutes at 15-lbs pressure. Under sterile conditions, each test material was placed in the well of a 12-well plate and exposed to a standardized bacterial suspension (2 mL of fresh broth and 20 µL of cell suspension) in brain heart infusion (BHI) broth followed by incubation at 37°C for 4 hours. Reference strains of P < 0.05.The data were analyzed using one-way ANOVA and Tukey post hoc tests. Significance was set at P < 0.05). Among these three, CH-GIC exhibited significantly higher values (P < 0.05). No significant difference was observed between the mean compressive strength values of C-GIC and MDPB-GIC (P > 0.05) (P < 0.05). Among the four modifications, MDPB-GIC exhibited the least flexural strength (P < 0.05)  . The mea < 0.05) .‒ release offers an advantage over other types as it determines only free ﬂuoride without interference from any type of covalent bond with other ions. At 24 hours, all the modifications showed significantly higher F‒ release than C-GIC (P < 0.05). Furthermore, BAG-GIC exhibited significantly higher F‒release than the other groups (P < 0.05)  . On day MDPB-GIC .S. mutans, 2.05 ± 0.1 OD and L. acidophilus, 2.2 ± 0.1 OD) compared to all the experimental groups, but less than the plastic mesh (negative control). The adhesion of S. mutans (0.04 ± 0.01 OD) and L. acidophilus (0.04 ± 0.008 OD) on MDPB-GIC was significantly less than all the other experimental cements and even less than the polystyrene strip that served as a positive control (0.09 ± 0.01 OD). CPP-ACP-modified GIC exhibited significantly less adhesion of S. mutans (1.5 ± 0.01 OD) and L. acidophilus (1.3 ± 0.09 OD) than BAG-GIC, CH-GIC, and C-GIC. Adhesion of S. mutans (1.5 ± 0.1 OD) and L. acidophilus (1.3 ± 0.03 OD) on BAG-GIC was significantly less than C-GIC. CH-GIC showed significantly less adhesion of S. mutans (1.7 ± 0.06 OD) and L. acidophilus (1.4 ± 0.01 OD) than C-GIC  in C-GIC. This could have reduced the interfacial tension between the glass-ionomer components, improving mechanical performance.This could be the reason why the carboxylic acid groups present in long polymer chains of MDPB could have enhanced the formation of salt bridges, improving the flexural strength of the modified GIC in the present study, despite a reduction in the volume of PAA by half. However, the mechanism beneath this inference needs to be studied in future experiments.‒ neither takes part in the acid‒base setting reaction of GIC, nor is it incorporated into the matrix structure.11 The highest F‒ release in BAG-GIC at 24 hours could be attributed to the higher solubility and dissolution rate of hydroxycarbonate apatite , which is formed on the surface of the BAG in an aqueous medium.13 Similar to BAG, the incorporation of CH might have had a catalytic effect, facilitating the diffusion of F‒ through the cement matrix towards the external medium. Additionally, the release of F‒ ions from the inorganic matrix seems to be favored when reinforced complexes have been formed.4 Reynoldsreported that the release of F‒ ions from CPP-ACP-modified GIC was promoted by the formation of CPP-ACP nanocomplexes.14 The same mechanism could have resulted in an increased F‒ release in CPP-ACP-GIC compared to C-GIC at both time intervals in the current study. MDPB-GIC showed no significant difference in its F‒ release when compared to C-GIC at both time intervals. Imazatoet al added MDPB to composite resin and reported that MDPB had an immobilized alkylpyridinium part, which was entrapped in the composite resin matrix.10 A similar entrapment could have happened within the solid glass matrix of GIC, with no interference with its F‒ release.F‒ releasing restorative materials.14 Hence in this study, the adhesion of S. mutansand L. acidophiluson the different modifications of GIC was evaluated using in vitro adhesion tests. In the present study, the cements were in contact with the bacterial inoculum for 4 hours. This period was selected because complete biofilm formation in the oral cavity usually occurs in 2‒4 hours.15Despite numerous studies on the morphology of oral biofilm and its effect on the tooth surface, only limited information is available on bacterial adhesion to restorative materials, especially on the surface of F16 In MDPB-GIC, the strong bactericidal action of MDPB is due to its cationic binding to the bacterial cell wall, which disturbs the membrane integrity, subsequently leading to leakage of cytoplasmic material and bacterial cell lysis.10 The polymer chains of MDPB could also have decreased the surface free energy of GIC, resulting in significantly reduced bacterial adhesion.17 Reynolds showed that the adsorption of CPP-ACP nanoparticles on the surface of enamel increases the surface net negative charge of enamel, influencing the long-term interactions with microbes through the development of repulsive forces.14 CPP-ACP has been shown to delay biofilm formation by preventing cell-to-cell adhesion of bacteria. These nanoparticles can also modify the long-term adhesion of streptococci by masking the streptococci-related receptors on salivary molecules.18 In BAG-GIC, the surface reaction of BAG with an aqueous medium produces an alkaline solution (pH = 9.8) that can kill the target bacteria. Thus even before a direct contact of bacterial cells with the BAG, they could be killed, reducing cell viability.19 CH-GIC exhibited significantly less adhesion. This could be attributed to the cationically charged amino groups of CH, which combine with anionic components, such as N-acetylmuramic acid, sialic acid, and neuraminic acid on the bacterial cell surface, resulting in impaired ion exchange with the medium, chelation of transition metal ions, and enzyme inhibition.20 This suppresses bacterial adhesion and growth.The increased surface roughness of GIC, coupled with the loss of surface integrity associated with fluoride release, predisposes C-GIC to higher bacterial adhesion.‒ release and bacterial adhesion of test materials, the literature shows that little or no correlation exists between the two factors. Hence, despite F‒ release, secondary caries ensues beneath and at the margins of the restorations.21 Among the experimental groups tested, BAG-GIC, CPP-ACP-GIC, and CH-GIC exhibited higher F‒ release, but the adhesion of bacterial strains to these experimental restorative materials was much higher compared to MDPB-GIC, which showed the least F‒ release among the experimental groups.While comparing the FThe various parameters in this study were tested under controlled in vitro conditions. Caution should be exercised to extend these results to the clinical settings, where the interplay of multiple factors and complex environmental changes occur. Future studies should assess the biocompatibility, mixing time, setting time, setting reaction, and adhesive properties of these experimental GIC formulations before their effective clinical use. Their durability in stress-bearing areas and individuals with high caries risk also need to be studied.‒ release but improved the resistance of C-GIC to bacterial adhesion.The incorporation of CPP-ACP, BAG, and CH improved the compressive and flexural strengths of C-GIC. The incorporation of MDPB did not adversely affect the mechanical properties and FConceptualization: NK and SV. Experimental work: NK. Original draft preparation: NK, SV, VS, SM, and RS. All the authors have read and agreed to the publication version of the manuscript.The preparation part of MDPB was guided by Dr. B. Karthikeyan PhD., the Department of Chemistry, Chidambaram, Tamil Nadu.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors deny any actual or potential conflicts of interest related to the study.Not Applicable.


This study aimed to characterize a cohort of patients with a refractory course of COVID-19, and to investigate factors associated with the duration of viral clearance (DoVC).A total of 65 patients with refractory COVID-19 were retrospectively enrolled from Huoshenshan Hospital. Univariate analysis and multivariate analysis were performed to examine the potential association between clinicopathologic characteristics and the DoVC.p = 0.002), serum level of aspartate aminotransferase (AST) > 21.8 IU/L , and titer of SARS-CoV-2 IgG > 142.09 AU/ml  were the three independent risk factors associated with delayed viral clearance.The median DoVC in the overall study cohort was 48 days (ranged from 21 to 104 days). Multivariate analysis indicated that fever at illness onset (Hazard ratio (HR) = 4.897, 95% CI [1.809–13.260], The current study suggested that clinical characteristics such as fever at illness onset, a high serum level of AST or SARS-CoV-2 IgG were associated with delayed viral clearance. Patients with these characteristics might need a more individualized treatment strategy to accelerate their recovery from the refractory COVID-19. As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sweeps across the globe, there is rising concern that the patients who recovered from COVID-19 may be at risk of reinfection. It was reported that a small percentage of patients were tested positive again after recovery from a previous episode of SARS-CoV-2 infection . HoweverThe median duration of viral shedding was reported to be about 20 days in many studies . HoweverIn our study, we characterized a cohort of patients who were deemed having a refractory course of COVID-19, with the purpose to identify the clinicopathologic risk factors associated with the delayed viral clearance.A total of 65 inpatients with refractory COVID-19 from Huoshenshan Hospital between February 8, 2020 to March 31, 2020 were enrolled in this retrospective study. All patients had a calculated DoVC longer than 20 days and had a previous history of COVID-19-related hospitalization before admitted to Huoshenshan Hospital. Patients with a calculated DoVC ≤ 20 days were excluded from this study. The severity of disease was evaluated based on the sixth version of Chinese clinical guideline for COVID-19 . Mild caA standardized sheet for data collection was used to retrieve the demographic, clinical, laboratory and treatment information from electronic medical records. Given the relatively high rate of false negative nucleic acid testing by nasal/pharyngeal swabs in the previous studies , we raisRespiratory samples were tested for SARS-CoV-2 RNA by the real-time reverse transcriptase polymerase chain-reaction (RT-PCR) test , following WHO guideline for qRT-PCR . SARS-CoAll patients were provided with necessary supportive care during the first and second hospitalizations, including but not limited to oxygen, antipyretic, antitussive, expectorant, antidiabetic, and antihypertensive agents. For etiological treatment, antivirals, antibiotics, traditional Chinese herbal tea or Chinese patent medicine were used either separately or in combination.p values ≤ 0.1 were included in multivariate analysis. Subsequently, log minus log function was used to identify the covariates which did not satisfy the proportional hazard assumption, and these covariates were excluded for the subsequent multivariate analysis. Multivariate analysis was conducted using Cox regression model to identify independent risk factors of prolonged viral clearance. Cases with missing data were excluded from the Cox regression model. A sensitivity analysis was performed to assess the robustness of the results , while data of laboratory results were presented as median . The medians of continuous variables were used as cut-off values to separate the patients into two groups for the sake of comparison, except for C-reactive protein and procalcitonin, where the upper limits of normal range were used. Univariate analysis was performed by using log-rank test to evaluate the association of patient characteristics with DoVC. All variables with ults see , S2. FinThe mean age of patients with a refractory course of COVID-19 was 52.5 years old, and 40 out of 65 patients were male. According to the sixth version of Chinese clinical guideline for COVID-19 , 51 out Complete blood count and blood biochemistries such as inflammatory seromarkers, renal, cardiac and hepatic function tests were measured upon admission to Huoshenshan Hospital . Of the p = 0.03), shortness of breath (p = 0.03), and fatigue (p = 0.03) at onset, as well as serum levels of CRP (p = 0.01) and SARS-CoV-2 IgG (p = 0.01) (p < 0.001), indicating an acceptable goodness-of-fit of the current model. Sensitivity analyses suggested no statistically significant difference in baseline and clinical characteristics between overall study cohort and patients included in Cox model , and titer of SARS-CoV-2 IgG > 142.09 AU/ml  were the three independent risk factors associated with longer viral clearance in the refractory COVID-19 patients , with a titer of SARS-CoV-2 IgG higher than 142 AU/ml . Patients with a serum level of AST > 21.8 IU/L were deemed to have only a statistically boundary significant longer DoVC .In univariate analyses, several clinicopathologic characteristics were found to be associated with the DoVC, including fever ( = 0.01)  and 2. Todel see , S2. Mulpatients . The propatients . The estThe DoVC is central for decision-making of nosocomial isolation precaution and post-discharge quarantine. Due to the high infectivity of SARS-CoV-2 and the overloaded medical system, a refractory clinical course of COVID-19 could be rather annoying for both patients and medical workers. However, there is still a lack of clinical guideline to deal with this particular group of patients. The current study documented the demographical, epidemiological, laboratory, and many other clinical data in a cohort of patients with a refractory course of COVID-19, with the purpose to identify the risk factors of prolonged hospitalization or viral clearance in these patients. In this retrospective study, 11 out of 65 (16.9%) were considered as possible reinfection , given te.g., anal swab or stool sample) after negative conversion of nasopharyngeal RT-PCR test (Before the cross-study comparison, it is worth noting that the definition of duration of viral shedding or viral clearance has not been unified in the previous studies . In consPCR test .In the current study, the DoVC had a very wide range , which iAlthough less importantly, attention should also be paid to the symptoms such as shortness of breath, fatigue and the serum level of CRP  and 2. HAlthough this study is bolstered by its design and methodological strength, it is also limited by its retrospective nature and small sample size. Not all tests were performed and monitored in all patients during hospitalization, especially the serology testing for SARS-CoV-2. Therefore, patients with incomplete data were excluded from multivariable regression analysis, which limited its statistical power despite a sensitivity analysis had proved the representativeness of the included samples. The patients with multiple hospitalizations might potentially subject to recall bias in retrieving the treatment history. Additionally, we only enrolled patients with refractory COVID-19, therefore, the results and conclusion should be customized to this specific group of patients, which limits the generalizability of this study. Finally, the ability to associate clinicopathologic characteristics with DoVC might be limited by using a convenient instead of statistically determined sample size.In conclusion, clinical characteristics such as fever at illness onset and a high serum level of AST or SARS-CoV-2 IgG were associated with delayed viral clearance in patients with a refractory course of COVID-19. Patients with these characteristics might need a more individualized treatment strategy to accelerate their recovery from the refractory COVID-19.10.7717/peerj.12535/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12535/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.12535/supp-3Supplemental Information 3Click here for additional data file.


Escherichia coli. Here, we present evidence that lipocalin Blc may be involved in heme binding, trans-periplasmic transport, or heme storage. This conclusion is supported by a cocrystal structure, mass-spectrometric data, absorption titration, and in silico analysis. Binding of heme is observed at low micromolar range with one-to-one ligand-to-protein stoichiometry. However, the absence of classical coordination to the iron atom leaves the possibility that the primary ligand of Blc is another tetrapyrrole.Lipocalins are a superfamily of functionally diverse proteins defined by a well-conserved tertiary structure despite variation in sequence. Lipocalins bind and transport small hydrophobic molecules in organisms of all kingdoms. However, there is still uncertainty regarding the function of some members of the family, including bacterial lipocalin Blc from  Heme is an essential molecule for aerobic organisms. Heme-containing proteins are crucial for many vital biological processes. This includes classical oxygen storage and transport, electron transfer, and catalysis, as well as more recently discovered roles in transcriptional gene regulation and ion channel regulation, among others .Lipocalins are a superfamily of functionally diverse proteins with remarkably conserved tertiary structure despite significant sequence variation. These ~ 20 kDa proteins form an eight-stranded antiparallel β-barrel enclosing an internal ligand-binding pocket. Lipocalins bind and transport small hydrophobic molecules in organisms of all three kingdoms of life. These include, but are not restricted to, fatty acids , heme 44,5, and 1-microglobulin. Nitrophorins are salivary proteins found in several blood-sucking arthropods. Nitrophorins use their prosthetic heme molecule to bind and transport nitric oxide (NO). NO induces vasodilation when released in a host organism upon biting. Nitrophorins are also shown to bind histamine, reducing inflammation and immune response elicited by the insects’ salivary proteins init are initial concentrations of a ligand and a protein, and Kd is the dissociation constant. Then, the total absorption at 411 nm (A411) of the protein–ligand solution in equilibrium can be calculated as follows:[PL] and ϵ[L] are extinction coefficients of, correspondingly, the protein–ligand complex and the free ligand at 411 nm at that concentration.Assuming 1 : 1 protein:ligand binding stoichiometry, the protein–ligand complex concentration in equilibrium ([PL]354) of the protein–ligand solution in equilibrium can be calculated as follows:[PL] and I[P] are fluorescence intensities of, correspondingly, the protein–ligand complex and the free protein at 354 nm when excited at 290 nm.The total fluorescence intensity at 354 nm  was crystallized at 21 °C in 1.6 M ammonium sulfate, 0.1 M MES, pH 4.5 with protein to precipitant volume ratio of 1 : 1 or in 1.6 M ammonium sulfate, 0.1 M MES, pH 4.5 supplemented with 0.1 M Iron(III) chloride hexahydrate or 5% w/v n-Dodecyl-β-D-maltoside according to the Hampton Research Additive Screen protocol using the sitting drop vapor diffusion technique. Crystals grew within 1 week and were flash frozen in liquid nitrogen using Parabar 10312 oil as cryoprotectant.WtBlc-split  are based on the common atom coordinates in the crystallographically determined pose of heme in complex with wtBlc-split fit with a maximum common substructure alignment . Split sFollowing relaxation, we performed local docking-based refinement of wtBlc:HP with RosettaLigand. Small molecule conformers were pregenerated with the BioChemical Library (BCL) cheminformatics toolkit , and 200http://biophysics.cs.vt.edu/H++) Second, we used absorption spectroscopy. During our work, we discovered that the recombinant protein expression in minimal medium  results in much less-colored protein samples , inset. Kd of 1.4 ± 0.3 μM. This value is comparable to those determined for full-length wtBlc binding to fatty acids [1-microglobulin [Kd on the order of 10−14 M) [Kd on the order of 10−12 M) [Kd on the order of 10−10 M) [After confirming our initial hypothesis of the presence of heme in the binding pocket of the wtBlc-split protein, we proceeded with a more detailed characterization of the discovered interaction. The heme molecule occupied the same hydrophobic cavity of the lipocalin as it was previously showed for vaccenic acid  Fig. S3Fig. S3. ty acids  or some globulin , bacteriglobulin , or someglobulin . The aff10−14 M) , some ot10−12 M) , or hemo10−10 M) .To further ensure that splitting of wtBlc did not fundamentally affect its ability to bind to heme and, therefore, that we can extrapolate our wtBlc-split:heme complex-based findings onto the full-length protein, we also performed the same spectrophotometric heme titration of the wtBlc-split protein. This titration yielded a similar dissociation constant of 2.0 ± 0.3 μM .Rhodnius prolixus (PDB ID 4XMC) [We then compared the heme binding and coordination by wtBlc-split with other known structures. We downloaded all available structures of lipocalins cocrystalized with heme from the PDB. All 67 of these belong to the nitrophorins family. While all proteins superimposed relatively well (~ 4 Å rmsd over backbone atoms), position of heme in the binding pocket in wtBlc-split is strikingly different from all other analyzed heme-binding lipocalins . Taking ID 4XMC) , which hIn the binding pocket formed by chains E and F of the structure, there are 19 amino acid residues containing atoms within a 4.0 Å distance from the ligand . Phe53 and Trp139 form π-stacking interactions with the porphyrin rings IV and II. The side-chain hydroxyl of Tyr137 and backbone amide of Gly113 stabilize one of the propionate groups of heme by hydrogen bonds. All other listed amino acids contribute to the hydrophobicity of the binding pocket . Heme alWhile the presence of heme propionate anchors as well as the described above heme ‘face’ and heme edge interactions are standard interactions that were previously reported in other heme-binding proteins , the claHowever, wtBlc may not have evolved specificity toward heme binding and may bind other porphyrins. To test this hypothesis, we evaluated the ability of wtBlc to interact with other chemically similar porphyrins: protoporphyrin IX, and non-natural porphyrins HP and verteporfin .Kd in the low nM range, but the sensitivity limitations of the method did not allow for accurate Kd determination. For more precise Kd determination, we exploited the tryptophan fluorescence quenching assay that was previously used for potential Blc ligand screening [Kd of 0.14 ± 0.1 nM  HP contains no coordinated iron, and (b) the vinyl groups in heme are hydrated to alcohols in HP. Nevertheless, they are structurally similar porphyrins, and it is not immediately clear why HP has a substantially higher binding affinity to wtBlc than heme. To further investigate this observation, we performed To our surprise, HP consistently departed from its initial heme-like binding mode to rotate ~ 90°, orienting both of the propionate groups toward the solvent front. To identify the most frequently occupied HP-binding modes, we generated HMM from the simulations of the wtBlc:HP complex. Our HMM identified five HP-binding modes: one heme-like binding mode (state 1), three variants of the 90-degree rotated binding mode , and one intermediate binding mode (state 2) . The traThe porphyrin rings of heme and HP overlap, which results in involvement of generally the same set of amino acids into HP ‘face’ and HP edge interactions  as in thHP, as well as other porphyrins and their derivatives, is well-known for its photosensitizing properties . While pUsing LC-MS/MS and absorption spectroscopy, we confirmed copurification of the heterologously expressed wtBlc and wtBlc-split proteins with heme. This allowed us to assign the observed ‘heme-like’ density in the obtained wtBlc-split crystal structure as a heme molecule and to investigate wtBlc:heme interaction in more detail.Based on the spectrophotometric hemin titration experiments and crystal structure analysis, we assessed the wtBlc hemin-binding affinity to be in the low μM range with 1 : 1 ligand-to-protein stoichiometry. While most of the observed protein:heme interactions are similar to those found in other heme-binding proteins, wtBlc-split does not coordinate the iron of the heme molecule. This observation prompted us to test whether wtBlc binds any other heme-like molecules. Interestingly, while the iron-free protoporphyrin IX molecule seems not to interact with the protein according to our absorption spectroscopy experiments, additional hydration of the two vinyl groups as seen in HP not only rescues the binding, but also improves the affinity by approximately three orders of magnitude.Blc is a membrane-associated protein anchored to the outer membrane by a covalently bound N-terminal cysteine lipid, and it faces the periplasmic space ,13. BaseThe observed light sensitivity of the wtBlc:HP complex was out of scope of the current research and therefore was not closely investigated. However, further study might result in creation of new oxygen sensing indicators, genetically encoded light-induced sources of reactive oxygen species, or tags for correlative light and electron microscopy.SuppTable S1. Data Collection and Refinement StatisticsTable S2. Binding free energy estimates of wtBlc:HP in different HMM metastable states.Fig. S1. Absorbance of solutions with different concentrations of free hemin at 411 nm (A) or free HP at 410 nm (B), and fitted curves (black dashed lines).Fig. S2. Schematic representation of the design of the leucine zippers-containing (“split-Zip”) and leucine zippers-free (“split”) DiB-split proteins constructs.Fig. S3. Comparison of different proposed wtBlc ligands’ binding sites.Fig. S4. Heme coordination in the binding pocket of the wtBlc-split protein, side view.Fig. S5. Time-dependent rmsd of heme to its crystallographically determined pose.Fig. S6. Structures of (A) Heme B, (B) protoporphyrin IX, (C) hematoporphyrin, and (D) verteporfinFig. S7. Metastable states of wtBlc:HP distinguished by a hidden Markov state model (HMM).Fig. S8. HP transitions from the initial heme-like binding mode to an alternative stable pose.Fig. S9. Light-induced changes of the wtBlc:HP solution.


Bone regeneration is a potential technique for treating osteoporosis. A previous study reported that F-box and WD-40 domain-containing protein 2 (FBXW2) localized with osteocalcin in bovine periosteum after 5 weeks of explant culture. However, the osteoblastic functions of FBXW2 remain unclear. In this study, double-fluorescent immunostaining was used to investigate the potential role of FBXW2 and its relationship with osteocalcin.At day 0, FBXW2 was expressed in the cambium layer between the bone and periosteum, while osteocalcin was expressed in bone. After explant culture, changes in the periosteum were observed from weeks 1 to 7. At week 1, partial FBXW2 expression was seen with a small amount of osteocalcin. At week 2, a layer of FBXW2 was observed. From weeks 3 to 7, tube-like structures of FBXW and osteocalcin were observed, and periosteum-derived cells were released from the periosteum in areas where no FBXW2 was observed. Bovine periosteum-derived cells can form a three-dimensional cell pellet, because multilayered cell sheets are formed inside of the periosteum in vitro. It is shown that in results FBXW2 is produced in periosteal explants near sites where initial osteogenic activity is observed, suggesting that it may be involved in periosteal osteogenesis.The online version contains supplementary material available at 10.1186/s13104-021-05825-z. Osteoporosis and bone fracture can decrease the quality of life of patients. Bisphosphonates are used for the treatment of osteoporosis, but they are associated with the risk of osteonecrosis of the jaw . As an aJapanese Black Cattle were raised in cattle farms for 30 months and slaughtered at the slaughterhouse for meat products . Bovine legs were chosen by staff at the slaughterhouse, not by researchers. Explant culture started within 24 h after the animal’s death. This study used dead bovine legs, and no treatment or experimental intervention for living animals was performed.The periosteum was separated from the bovine leg as described previously . The obsAll paraffin sections were pretreated with ready-to-use Proteinase K  for 10 min for antigen retrieval. The primary antibodies used were mouse anti-bovine osteocalcin monoclonal antibody  and goat anti-FBXW2 polyclonal antibody . The secondary antibodies used were goat anti-mouse Alexa Fluor™ 488-labeled antibody , mouse anti-goat IgG-CFL 594 , and N-Histofine Simple Stain AP . The alkaline phosphatase-tagged antibodies were visualized with PermaRed/AP . Incubation with the anti-bovine osteocalcin monoclonal antibody (diluted 1:500) and anti-FBXW2 antibody (diluted 1:100) was performed for 4 h at room temperature. Cell nuclei were stained with hematoxylin or 4′,6-diamidino-2-phenylindole (DAPI). For the negative controls, an antibody against the receptor activator of NF-κB ligand  and normal goat serum  were used. Images were obtained using a fluorescence microscope  and BZ-II Viewer  and BZ-II Analyzer  software. One researcher performed all stages of the experiments and data analysis.FBXW2 was expressed in the cambium layer of the periosteum, but showed little expression in bone .The alkaline phosphatase-labeled antibody against osteocalcin that was used in the present study was the same as that used in a previous study ; regardlThe periosteum consists of a cambium layer and a fibrous layer in vivo. The cambium layer is osteogenic and the fibrous layer is poorly osteogenic. In this study, FBXW2 and osteocalcin were in contact with each other in vivo .Additional file 1: Fig. S1. Schema of this study. a At day 0, the periosteum, cambium layer, and bone were observed. b From weeks 1 to 7, changes in the periosteum were observed in explant cultures.Additional file 2: Fig. S2. Fluorescent immunostaining and immunohistochemistry of bone at day 0. FBXW2 is expressed in the cambium layer, and osteocalcin is expressed in bone. Scale bar: 100 μm. (a) FBXW2: red. (b) Negative control for (a). (c) Osteocalcin. (d) RANKL: negative control for (c). (e) Osteocalcin: green. (f) RANKL: negative control for (e). P: periosteum; B: bone; LOB: lacuna of bone.Additional file 3: Fig. S3. Synthesis of FBXW2. Double-fluorescent immunostaining of the periosteum at day 3 showing periosteal cell synthesis of FBXW2 and tube-like structures of FBXW2 bursting out of the cells. Scale bar: 100 μm. Osteocalcin: green; FBXW2: red; DAPI: blue.Additional file 4: Table S1. Changes in the expression of FBXW2 and osteocalcin.


Silicon nanoparticles (SiNP) are currently of great interest, especially in biomedicine, because of their unique physicochemical properties combined with biodegradability. SiNPs can be obtained in various ways and can have either a non-porous solid (sol-) or porous (por-) structure. In this work, we carry out detailed optical monitoring of sol- and por-SiNP biodegradation using Raman and photoluminescence (PL) micro-spectroscopy. SiNPs were obtained by ultrasound grinding of sol- or por-silicon nanowires, created by silver-assisted chemical etching of crystalline Si with different doping levels. In this case, sol-SiNPs consist of nanocrystals 30 nm in size, while por-SiNPs consist of small 3 nm nanocrystals and 16 nm pores. Both SiNPs show low in vitro cytotoxicity towards MCF-7 and HEK293T cells up to 800 μg/mL. The appearance of the F-band (blue–yellow) PL, as well as a decrease in the intensity of the Raman signal, indicate the gradual dissolution of the sol-SiNPs during 20 days of incubation. At the same time, the rapid dissolution of por-SiNP within 24 h is identified by the quenching of their S-band (red) PL and the disappearance of the Raman signal. The obtained results are important for development of intelligent biodegradable drug delivery systems based on SiNPs. Both However, improper usage of SiNPs may be dangerous, i.e., SiNPs may potentially agglomerate, facilitate blood clots formation, interfere with cell mitosis; excessive formation of silicic acid may significantly change pH value ,11. TherThe gold standard method is inductively coupled plasma atomic emission spectroscopy (ICP-AES), which gives precise amount of Si in supernatant of centrifugated SiNPs suspension ,13. ThesThe optical methods allow to estimate both Si amount and average size of Si nanocrystals (nc-Si), which forms por-SiNPs. In general, size of nc-Si can be determined by position of both Raman and PL specific bands ,18. MoreSiNPs can be obtained from big variety of silicon-based materials and demonstrate different properties. However, all of them can be considered as por-SiNPs or sol-SiNPs, therefore that two types of nanomaterials have been chosen for present articles. Here, we utilized SiNPs obtained from Si nanowires created by metal-assisted chemical etching of crystalline Si wafers with different doping levels .The present article shows how dual, Raman and luminescent, noninvasive optical monitoring of SiNPs biodegradation via their physicochemical characterizations on the different stages of that process can be made for two most important cases: porous or solid silicon-based nanoparticles.Both sol-SiNPs and por-SiNPs were obtained by procedure schematically shown in 3 and 5 M of HF in a volume ratio of 1:1 for 15 s. As a result, Ag nanoparticles (AgNPs) were sedimented on the surface of the c-Si wafer (shown as gray circles). Then, the c-Si wafer was placed into a mixture of 5 M HF and 30% H2O2 in the volume ratio 10:1 for 1 h. The layer of SiNWs occurred because of Ag nanoparticles-assisted chemical etching. Then, the samples were rinsed 3 times in bidistilled water and dried at room temperature. Etching of highly-doped (HD) c-Si with resistivity of 0.001 Ohm*cm resulted in formation of porous nanowires, while etching of low doped (LD) c-Si with resistivity of 1 Ohm*cm leads to formation of solid (non-porous) nanowires. That property of SiNWs is inherited by the resulting SiNPs.As the first step of MACE, a (100)-oriented single crystalline silicon wafer was immersed in a mixture of 0.01 M AgNO3. Then, Si wafers with the SiNWs is subjected to ultrasonic treatment for 12 h, which results in detaching of the nanowires from the substrate and their fracturing into nanoparticles with a low aspect ratio. Finally, after several centrifugation steps, SiNPs aqueous suspension is acquired.Then, after the SiNW layer is formed, AgNPs are removed by dissolution in HNO2 adsorption/desorption (Micromeritics Tristar 3000). The pore area from the adsorption branch using BET theory , and the pore size was calculated from the desorption branch using BJH theory . Before measurements, the samples were dried by lyophilization. Fourier transform infrared (FTIR) spectra were measured by using Bruker spectrometer and attenuated total reflectance (ATR) addon.Structural analysis of the samples was carried out by Carl Zeiss ULTRA 55 scanning electron microscopy (SEM) for SiNW layers and by LEO912 AB OMEGA transmission electron microscopy (TEM) for SiNPs. The samples of NPs for TEM studies were prepared by deposition of the nanoparticle powder on the standard carbon-coated gold TEM-grids. TEM images were processed with ImageJ Software to obtain the silicon nanocrystals (nc-Si) size distribution. To determine the hydrodynamic size of the nanoparticles a Malvern Instruments Mastersizer 200 have been used. The specific surface area of the SiNPs as well as their pore sizes and pore volumes were determined with NSiNPs were introduced into phosphate-buffered saline  to achieve the concentration of 0.5 mg/mL, then 5 mL of nanoparticles suspension were placed in dialysis bag (4.5 kDa pore diameter) and then in 200 mL of PBS at 37 °C (Binder chamber).To measure the photoluminescence (PL) or Raman spectra, 0.1 mL of the suspension was dried on a metal plate after different time intervals of the incubation. The PL spectra of the samples were measured under excitation with a cw Ar-ion laser at 364 nm . The PL signal was detected using a grating monochromator  equipped with a CCD array. Raman spectra were measured using a Confotec™ MR350 confocal Raman microscope with laser excitation at 633 nm and a weak power of 1 mW to protect samples from overheating. MagicPlot software was used for deconvolution of spectra.2 at 37 °C, in DMEM  medium containing 4.5 g/L glucose (Gibco), 10% FBS (Gibco), in the presence of a mixture of antibiotics and antimycotics (Gibco). All cells were maintained in the logarithmic growth phase for experiments. After 24 h, the culture medium was replaced with fresh one for treatments. Before the treatment with nanoparticles, staurosporine or MG132  the culture medium was removed, and cells were washed with PBS. Fresh serum containing medium  was added to cells. All cells were treated with nanoparticles (por-SiNPs and sol-SiNPs) in concentrations of 400 and 800 µg/mL and incubated for 24 h. Additionally, MCF-7 cells were treated with staurosporine in concentration 0.25 µM (4 h) and HEK293T cells were treated with MG132 in concentration 2 µM (24 h). Both agents were used as inductors of apoptosis.The studies were performed using breast carcinoma MCF-7 cells and human embryonic kidney HEK293T cells, which were kindly provided by the Departament of Toxicology Karolinska Institutet . All cells were grown in 5% CO5 cells (10 μL) were transferred into 200 μL of 1× annexin-binding buffer  and 2 μL of Annexin V-FITC (Invitrogen) was added. Then, samples were incubated in the dark at room temperature for 15 min. Next, 5 μL of propidium iodide (50 μg/mL) (BD Biosciences) was added to each sample. The analysis of the cell population after 5 min of incubation in the dark at room temperature was performed using a BD FACS Canto II flow cytometer (BD Biosciences).After the indicated time of treatment, cells were detached from the dishes using 0.25% trypsin (Gibco) and transferred into a conditioned medium. Then, cells were centrifuged , washed twice with cold PBS solution , and the pellet was resuspended in PBS (100 μL per 1 million cells). Approximately 10g, 15 min, 4 °C), a part of the supernatant was taken for protein concentration assay using the Pierce BCA Protein Assay Kit . Then, samples were mixed with Laemmli’s loading buffer and boiled for 7 min. Equal amount (20–40 μg) of protein extracts was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis  at 100 mV, followed by blotting onto nitrocellulose membranes for 2 h at 110 V. After transferring, membranes were blocked for 1 h with 5% non-fat milk in tris-buffered saline (TBS) at room temperature and subsequently probed overnight with appropriate primary antibodies (1:1000), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (from 1:2500 to 1:5000) for 1 h with 2.5% non-fat milk in TBS at RT. Detection was visualized with ECL  and a ChemiDoc MP Imaging System .After the indicated time of treatment, cells were detached with scraper in the conditioned medium. The cells were centrifuged  and washed twice with ice-cold PBS (Paneco). Then, the cell pellet was lysed in RIPA buffer (25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, cOmplete™ Protease Inhibitor Cocktail ), and incubated on ice for 20 min. After centrifugation -polymerase , tubulin-alpha , anti-rabbit full and cleaved caspase-3 . Anti-rabbit or anti-mouse IgG, conjugated with horseradish peroxidase  were used as secondary antibodies.Confocal luminescent imaging experiments were performed by using a TCS SP5 Leica confocal laser scanning microscope with excitation at 405 nm. The SiNP PL signal was recorded in the range of 510 to 800 nm. An oil immersion Leica 40×/1.25–0.75 objective was used for imaging.Cells were grown on coverslips in a standard 10 cm Petri dishes filled with the DMEM culture medium. When cells reached the desired confluency, the media was removed from the dish and cells were washed with PBS. Fresh serum containing medium  was added to cells. All cells were treated with nanoparticles (por-SiNPs and sol-SiNPs) in concentration 800 µg/mL and incubated for indicated time: por-SiNPs—9 and 24 h; sol-SiNPs—24 h and 7 days. After selected time of incubation, all cells were washed two times for 3 min with PBS and were fixed in 4% paraformaldehyde solution in PBS during 7 h at +4 °C.2 slides aiming to prevent a background scattering. Cells were grown in DMEM culture medium. When cells have reached the desired confluency, the media was removed from the dish and cells were washed with PBS. Fresh serum containing medium  was added to cells. All cells were treated with nanoparticles (por-SiNPs and sol-SiNPs) in concentration 800 µg/mL and incubated for indicated time: por-SiNPs—6 and 24 h; sol-SiNPs—24 h and 7 days. After a selected time of incubation, all cells were washed two times for 3 min with PBS and were fixed in 4% paraformaldehyde solution in PBS during 7 h at +4 °C. Afterwards, the glasses with the cells were placed in standard 5 cm Petri dishes filled with distilled water. This prevents the cells from overheating during the measurements due to high laser power and enables longer imaging of the cell clusters. For this purpose, a 63×/NA 1.0 water dipping objective Zeiss was used to focus the laser on the cell. The obtained images of cell and intracellular uptake of nanoparticles were generated by applying a CCD camera Andor operating at −60 °C.Micro-Raman spectroscopy data were acquired using a confocal Raman microscope Confotec™ MR350, with laser excitation at 633 nm wavelength and 0.5 mW power. The MCF-7 and HEK293T cells were fixed on special CaF20 free holes, which are necessary for the formation of pores inside the nanowires during the etching process. Therefore, porosification affects the whole volume of HD nanowire, in contrast to LD nanowire (LD p-type c-Si contains of ~1015 free holes), where only surface is partially etched  and low-doped (LD) c-Si substrate, correspondingly. The structural difference between two types of nanowires can be explained by the doping level of the c-Si substrates used . HD p-tyy etched . After uThe obtained TEM images of sol-SiNPs and por-SiNPs are shown in TEM images allow estimating the typical size of SiNPs, which is in the range of 100 to 300 nm and corresponds to the DLS measurements C,F. DiffDark-field TEM image of SiNPs tuned to a certain diffraction peak  was usedAdditionally, nitrogen gas adsorption/desorption analysis was used to determine the specific surface area, pore volume, and pore diameters of the obtained samples . Large v−1, which is attributed to thick oxide layer at the surface of SiNPs [3 treatment aimed to dissolve Ag nanoparticles inside Si wafers after MACE-process. Therefore, both samples are well oxidized, which provides them good hydrophilic properties and similar surface composition.FTIR spectra of both sol-SiNPs and por-SiNPs are shown in of SiNPs . Mostly,SiNPs exhibit unique optical properties, which were used for monitoring of their dissolution process. First of all, the modeling experiments of SiNPs incubation in dialysis bag in PBS at 37 °C were carried out A. The exThe high-energy band (2.3 eV), which is slightly pronounced in PL spectrum of initial sol-SiNPs, dramatically increases with incubation time. It is attributed to an amorphous intermediary layer, which can be considered as a shell of big non-fluorescent nc-Si. Changes of PL intensity show that this layer is negligible in initial sol-SiNPs, then grows during the incubation (~20 days), and finally starts to dissolve after the whole Si core is converted into amorphous phase (after 20 days).Similar processes take place with por-SiNPs, i.e., reduction of excitonic band and enhancement of defect band. However, por-SiNPs structure is different: they are consisted of much bigger amount of small nc-Si with quantum confinement and there is no big nc-Si suitable for growing of thick amorphous layer. Therefore, the low-energy band (1.7–1.8 eV) vanishes along with dissolution of small nc-Si, but some of them nevertheless convert into amorphous nanoparticles and contribute to high energy bands (2.5–3.5 eV). Note that full decomposition of typical PL spectra of por-SiNPs is shown in All excitonic bands of PL can be used for estimation of average nc-Si diameter in the samples. According to quantum confinement models  peak pos−1. Intensity of this line is proportionate to the volume of scattering Si. Nanocrystalline Si (with nc-Si diameter < 7 nm) is characterized by a similar line but broadened and shifted depending on nc-Si sizes and dispersion of nc-Si ensemble [Raman spectra also allow monitoring of SiNPs biodegradation. Crystalline silicon exhibits narrow Raman band at 520.5 cmensemble . Accordi−1 corresponding to light scattering in amorphous silicon. The diameter of nc-Si in por-SiNPs, calculated from the position of the Raman spectra maximum [In the Raman spectra of por-Si nanoparticles E, after  maximum  was abouTherefore, both Raman and PL spectra show that typical dissolution time is about 15–21 days for sol-SiNPs and 24 h for por-Si. The difference is explained by different nc-Si sizes, and also their different specific surface area which is an important factor dissolution rate .To study whether nanoparticles (por-SiNPs and sol-SiNPs) in concentration 400 and 800 µg/mL cause death of human embryonic kidney HEK293T cells (shown below) and breast carcinoma MCF-7 cells shown in , WesternA broad-spectrum protein kinase inhibitor, Staurosporine (STS), in concentration 0.25 μM for 4 h and a proteasome inhibitor MG132 in concentration 2 μM for 24 h were used as positive controls for apoptosis induction in MCF-7 and HEK293T cells, respectively. MG132 induced activation of effector caspase-3 and PARP cleavage in HEK293T cells, and STS caused cleavage of PARP in MCF-7 cells. Both agents decreased cell viability to 77% and 74% compared to non-treated cells in MCF-7 and HEK293T cells, correspondingly .−1. The next image shows that after 7 days of incubation, the concentration of sol-SiNPs is much smaller and the Raman signal is weaker. However, it is still clearly pronounced, and no shift of the band is detected; this is consistent with previous results obtained for PBS. Note, that internalization of SiNPs inside the cells was verified by simultaneous presence of both nc-Si band and cell components bands (mostly by proteins and lipids) in the Raman spectra  can be present, according to PL spectra see . Such smLuminescent imaging also allows to visualize SiNPs in cells. However, the method is better applicable to por-SiNPs due to the larger amount of tiny luminescent nc-Si in them than for sol-SiNP. Sol-SiNPs internalized after 1 day exhibit weak luminescence, therefore, they are visible only by adsorption on widefield images. However, after 7 days of incubation with cells, PL increases and sol-SiNPs become more visible in fluorescence mode.On contrary, por-SiNPs are in a great abundance inside cells cytoplasm just after 9 h of incubation. After 24 h, almost complete biodegradation of por-SiNPs occurs and their PL signal is much weaker, as it is seen in The important question for correct interpretation of the obtained results is verification of successful internalization of SiNPs inside cells. As it may be difficult to see internalization directly in presented Thus, two optical noninvasive methods of monitoring of SiNPs biodegradation were developed. They were applied to two types of nanoparticles, i.e., solid and porous nanoparticles, which can be considered as reference materials for all variety of silicon-based nanomaterials. Both samples were prepared by similar etching procedures of low- and high-doped Si wafers in order to minimize influence of other factors. Ultrasonic grinding of the obtained Si nanowires formed 140 nm sol-SiNPs and 130 nm por-SiNPs consisted of 30 nm and 3 nm Si nanocrystals, respectively.Structural differences lead to optical ones. Por-SiNPs were found to be contrast in luminescent bioimaging, while sol-SiNPs have better contrast in Raman spectroscopy. This was explained by quantum confinement effects and high porosity of por-SiNPs, which resulted in efficient S-band PL in red range, originated from excitons confined in small nanocrystals. A similar band was found for sol-SiNPs, but it vanished after 1–2 h of incubation and attributed to minority of small nanocrystals on the surface of the nanoparticles. Further luminescent properties of sol-SiNPs are attributed to F-band (blue–yellow range) PL originated from defects in emerging oxide shell during their biodegradation. The optical study showed that typical biodegradation lifetimes were 20 days for sol-SiNPs and only 24 h for por-SiNPs. This difference is caused by different nc-Si sizes, which is proportionate to the biodegradation rate. The study included SiNPs dissolution in dialysis bag in phosphate buffer saline at 37 °C  and spectral-resolved Raman measurements and photoluminescent bioimaging of SiNPs inside HEK293T and MCF-7 cells. Both experiments showed similar results.Both types of SiNPs exhibited no toxicity towards MCF-7 and HEK293T cells in concentration up to 800 μg/mL. Western blot analysis revealed that both por-SiNPs and sol-SiNPs did not led to a generation of caspase-3 p17/19 active fragment and subsequent cleavage of its substrate–PARP, which pointed to the absence of necrosis and apoptosis. These results were supported by flow cytometric analysis with double-staining method (Annexin V–FITC and propidium iodide (PI)), which also showed negligible population of apoptotic and necrotic cells after 1, 2, and 3 days of incubation.Thus, it is possible to tailoring the rate of biodegradation of nanoparticles by changing the size of their nanocrystals. Two complimentary optical methods can be used for biodegradation monitoring of the variety of silicon-based materials. Raman imaging is most applicable for those with big (>5 nm) nanocrystals, while photoluminescent imaging is suitable for materials with small (<5 nm) nanocrystals. The results obtained can be useful for the development of intelligent drug delivery systems based on silicon nanoparticles with a tailored biodegradation time for sustained drug release and the development of new approaches in theranostics of diseases.


The proposed sub-6GHz antenna covers the following frequency bands: 4–4.5 GHz, 3.1–3.8 GHz, 2.48–2.9 GHz, 1.82–2.14 GHz, and 1.4–1.58 GHz, while the millimeter-wave antenna operates at 28 GHz with at least 500 MHz of bandwidth. A complete antenna analysis is provided via a step-by-step design procedure, an equivalent circuit diagram showing the operation of the shared aperture antenna, and current density analysis at both millimeter-wave and sub-6GHz bands. The proposed antenna design is also characterized in terms of MIMO performance metrics with a good MIMO operation with maximum envelop correlation coefficient value of 0.113. The maximum measured gain and efficiency values obtained were 91% and 8.5 dBi over the entire band of operation. The antenna is backward compatible with 4G bands and also encompasses the sub-6GHz and 28 GHz bands for future 5G wireless communcation systems.A shared aperture 2-element multiple-input-multiple-output (MIMO) antenna design for 5G standards is presented in this study, one which uses the same radiating structure to cover both the sub-6GHz and millimeter-wave (millimeter-wave) bands. The proposed antenna comprises four concentric pentagonal slots that are uniformly separated from one another. For the sub-6GHz band, the antenna is excited by a single open-end microstrip transmission-line, while a 1 × 8 power divider (PD) connected via a T-junction structure excites the millimeter-wave band. Both the sub-6GHz and mm-wave antennas operate in a MIMO configuration. The proposed antenna design was fabricated on a 120 × 60 mm It is a major leap beyond the 4G network, as it offers many features which were not available before. In particular, it offers massive connectivity, low latency, high reliability, and much higher data rates than its predecessors. The new architectures of 5G will require major changes in hardware implementation when it is deployed. Unlike the previous standards, 5G systems, will also use millimeter-wave bands for the first time . This isThe adoption of frequency bands which are spaced widely apart has posed specific challenges in the design of the radio frequency (RF) front-end, especially in relation to the antenna. Most of the devices connected with the cellular system have a small form factor. This is particularly true for internet-of-things (IoT)- based devices, which most of the time have very a small size. An antenna design which can work for all these bands, or having multiple antennas covering individual bands, will require due consideration to meet the required radiation characteristics while easily fitting inside a given device.Multiple-input-multiple-output (MIMO) systems are an important technological breakthrough which can offer better channel capacity and reliability of communication within a limited bandwidth and transmission power. They have been adopted in several communication standards, including the 4G standard and WiFi, etc. MIMO systems will remain important in the context of the 5G standard as well . In the The antenna is a key component of a MIMO system, and its design has a significant impact on the overall system’s performance. Ideally, such an antenna is required to possess very high port isolation between its antenna elements while the radiation patterns of its elements are such that the MIMO channels are kept independent. The envelop correlation coefficient (ECC) is the performance metric of a MIMO antenna which determines how much correlation between channels is added as a result of the antenna. Practically, such a MIMO antenna is designed to have high port isolation between its antenna elements and low ECC values .2 FR4 substrate with a thickness of 1~mm. With a dual polarized operation, the antenna achieved a reasonable isolation of 12.5 dB between its antenna elements. In [2 area. The metal rim of a mobile phone was utilized to create an 8-element wideband MIMO antenna in [2. Similarly, a MIMO antenna design for 5G smartphone applications which covers the 3.5~GHz 5G band can also be found in [Lately, several MIMO antenna designs have appeared in the literature for 5G systems. They can be broadly categorized into two categories based on their frequency of operation. Many designs are only for the sub-6GHz band of 5G while other designs focus on the millimeter-wave bands. In the sub-6GHz bands, the design of MIMO antennas of up to ten elements can be found in the literature for handheld devices. The focus of many of the designs is to achieve multi-band or wideband characteristics with improved isolation in closely placed antenna elements. In , an 8-eltenna in . The antfound in ,10 whereThe antennas designed for 5G applications covering the millimeter-wave band are mainly focused on having an array with high gain and highly directive beams. Many of these designs are antenna arrays in which multiple radiating elements are used together to achieve the desired radiation characteristics. In , an arraIn addition to designs which incorporate arrays and single elements for arrays to achieve high gain in the millimeter-wave band of 5G, some MIMO antennas for 5G in the millimeter-wave band have also appeared in literature ,20. In , a quasiThe distinctive characteristic of the proposed antenna design is the integration of sub-6GHz with a millimeter-wave antenna that uses the same radiating aperture/structure.This study provides a unique concept for 5G access point applications based on a shared-aperture sub-6GHz and millimeter-wave concentric pentagonal slots-based antenna design. It eliminates the need for multiple antenna structures to be used for different operating bands, namely the sub-6GHz and millimeter-wave bands.The multi-band antenna design proposed in this work operates on several fundamental modes corresponding to each concentric slot. As a result, the antenna appealing due to the higher gains and efficiency values attained for every dominant mode.The MIMO performance of the proposed antenna design is well-suited to be operated in both the sub-6GHz and 28 GHz millimeter-wave bands.Despite their distinct feeding structures, the proposed design employs a co-shared radiating structure covering the desired sub-6GHz and millimeter-wave bands. This is one of the distinguishing traits of the proposed antenna design.The covered bands for sub-6GHz are 4–4.5~GHz, 3.1–3.8~GHz, 2.48–2.9~GHz, 1.82–2.14~GHz, and 1.4–1.58~GHz, whereas the millimeter-wave ranges from 27.8 GHz to 28.3 GHz with a minimum bandwidth (BW) of 500 MHz. The operation of such a hexa-band MIMO antenna for both sub-6GHz and millimeter-wave bands has not been reported for any antenna previously.This paper proposes a novel 2-element MIMO antenna design to be used for 5G-enabled access point devices, in which the same elements operate in the sub-6GHz and millimeter-wave bands, although with different feeding structures. The contributions of the proposed work are:The paper is organized as follows. In In this section, details about the geometry of the antenna, a circuit diagram, the theory behind the operation of the antenna, and the design procedure are discussed in detail.rε) of 3.48. The total size of the board used was 60 × 120 mm2. Each antenna element is comprised of four concentric pentagonal slots etched out from the ground (GND) plan. The gap between the slots and the width of each slot are 2 mm and 0.8 mm, respectively. The optimal spacing between the antenna elements was found to be 70 mm. The proposed slot-based shared aperture design was excited with an open-ended transmission-lines as well as with 1 × 8 PD. The transmission-lines (Feed-1 and Feed-2) and PD (Feed-3 and Feed-4) were used to excite the same slot structure for sub-6GHz bands and millimeter-wave band at 28 GHz, respectively. Feed-1 and Feed-2 were regular SMA connectors which were utilized for sub-6GHz operation, while Feed-3 and Feed-4 were PCB receptacle 2.92 mm F type connectors that cover up to 40 GHz. The fabricated model of the proposed antenna is shown in The shared aperture slot antenna proposed in this work was designed to cover as many sub-6GHz 5G bands as possible as well as the millimeter-wave band at 28 GHz. For the proposed work, a pentagonal shaped slot was selected due to the fact that it can achieve multi-band frequency characteristics and better input impedance matching compared to annular slot antennas . AdditioS11 plots for the two and three concentric pentagonal slots are shown in To achieve the lowest possible frequency bands, a single pentagonal slot was initially chosen. A single slot antenna with resonance curves, depicted in S11 curves for optimized design are depicted in In order to determine the antenna’s dimensions, its resonating bands, together with adequate input impedance matching and proper placement on the board, a number of parametric sweeps were performed. To achieve the desired performance, the width of each pentagonal slot and the spacing between them was optimized. The inZ. The length and width of the feed line were optimized in order to match the input impedance inZ. For sub-6GHz operation, parametric sweeps were performed on the length of the feed line  and the transmission curves Sn1, respectively, where n = 2, 3,…, 9, denotes the number of output ports  of the PD. It can be observed that between the frequencies of 27 GHz and 29 GHz, the input port transmitted an equal amount of power. This is the millimeter-wave antenna’s desired operating band.A 1 × 8 PD was designed at 28 GHz, as shown in S11 are shown in S33. A slight variation in scattering parameters was observed for both cases, as shown in To obtain the desired PD characteristic, numerous parametric sweeps were carried out. S11 curves are shown in To elaborate on the antenna’s operation, although the two antennas at the sub-6GHz and millimeter-wave bands share the same aperture, they are excited by a different set of transmission lines. Thus, the effective aperture of the two antennas is different and the millimeter-wave antenna is not of a higher order in terms of mode compared to the sub-6GHz antenna. We investigated further by simulating the proposed design while Feed-1 and Feed-2 were active with a frequency sweep from 27–29 GHz. The resulting The planar slot-based antenna design is an attractive choice for researchers because of its numerous advantages. Both ends of an open-ended and short-circuited slot-antenna, respectively, can be modeled as λ/4 and λ/2 transmission lines, corresponding to the resonating band fundamental mode , as giveRLC circuit to aid in the understanding of the shared aperture slot structure for the sub-6GHz and millimeter-wave bands. A microstrip slot-line transition represents an interesting option for the slot antenna feeding mechanism because of its simple planar structure, efficient coupling with the slot, and well-known behavior [The circuit model of the square slot-line sub-6GHz radiator is shown in behavior . The cirbehavior . In the lot-line ,29. The RLC circuit, as shown in the equivalent circuit model, while the inner two slots are also inductively coupled with each other. Similarly, for the millimeter-wave band antenna, the same antenna aperture was shared and acts as a connected slot array fed with Port-3. This can be modeled as a parallel resonating RLC circuit, as shown in From the design evolution process, it can be seen that each slot-line did not affect the previous resonating band. Each slot in the proposed sub-6GHz antenna structure operates at its fundamental mode of operation see a–c, resu™. Both the sub-6GHz and millimeter-wave bands were tested with the optimized antenna design. In this section, the measured and simulated S-parameters, gain patterns, and MIMO performance parameters are discussed in detail. The port parameters were obtained using an Agilent PNA, while a Satimo Starlab chambers were used for measurement of patterns.The proposed shared aperture MIMO slot antenna was designed and modeled using HFSSThe measured and simulated S-parameters of the proposed antenna design are presented in Similarly, the activation of Feeds 3 and 4 causes the antenna to function in the millimeter-wave band. The reflection coefficient when these feeds are excited is shown in The shared aperture MIMO antenna proposed in this work was characterized in terms of its far-field radiation patterns. The efficiency  and where the precise location of the receiver and transmitter are known, directional patterns are highly desirable. However, in a close proximity communication scenario, where the location of the receiver and transmitter are not known, MIMO antennas are more suitable, especially in a scattering rich environment ,29.The radiation pattern at 28 GHz is not directional, which was expected because of the slot-based antenna design. A simple modification was made for this design to reduce the ripples and make the patterns more directional by using a backing reflector, which is usually present because of the electronics behind the antenna in access point applications. A simulation was performed to show the patterns at 28 GHz. The correlation coefficient (en below :(2)ρe=|∬ECC values were computed for the sub-6GHz/millimeter-wave bands and are shown in DG) values were computed. In a MIMO system, higher values of signal to noise ratio (SNR) are obtained for uncorrelated signals and hence better signal reception is provided. Diversity gain is the difference between the time-averaged SNR of the given MIMO diversity antenna system and that of a diversity channel of a single antenna system with the same SNR reference level. Mathematically,DG values for sub-6GHz and millimeter-wave bands are given in To assess the proposed shared aperture MIMO antenna system’s diversity performance, diversity gain  can be used to characterize the frequency response of a MIMO antenna effectively. This can be defined as the ratio of the square root of the total reflected power to the square root of the total incident power and is given by expression blow:j0, while this varied from Case-1:Case-2:Case-3:Case-4:Case-5:For the proposed shared aperture MIMO antenna design, TARC curves for sub-6GHz/millimeter-wave bands were obtained. The phase of the first exciting port was kept at 1eMEG) accounts for the antenna coverage area and the achievable data rates of the MIMO antenna system. MEG can be calculated via the mutual relation between the field patterns of the antenna and the statistical distribution of signals in the urban environment [MEG of an antenna system is determined from its two-dimensional field patterns in a mobile wireless environment.MEG is −3 dB for urban environments with a 100% efficient antenna, while for practical designs, the value can be as low as −12 dB. The MEG of the proposed MIMO antenna was computed for sub-6GHz/millimeter-wave bands using Equation (5) and the values are provided in XPD.The mean effective gain (ironment . The MEGThis work presents a 2-element shared aperture MIMO antenna design for 5G-enabled devices and access point applications. This work can be extended by incorporating 4-elements within the same aperture with polarization diversity and enhanced gain. The shared aperture concept can also be implemented in mobile terminals and other wireless handheld devices.2 substrate board. The proposed sub-6GHz antenna covers the following frequency bands: 4–4.5~GHz, 3.1–3.8~GHz, 2.48–2.9~GHz, 1.82–2.14~GHz, 1.4–1.58~GHz, and 27.8–28.3~GHz, while the millimeter-wave antenna operates at 28 GHz with a minimum bandwidth of 1 GHz. The simulation results were in close agreement with the measured results. The antenna is not only backward compatible with 4G bands, but it also has coverage for 5G sub-6GHz and 28 GHz bands.This work has presented a 2-element shared aperture MIMO antenna design for 5G-enabled devices and access points. The design covered the sub-6GHz and millimeter-wave bands with the same radiating elements, and four concentric pentagonal slots comprised the single element. To excite the antenna for the sub-6GHz and millimeter-wave bands, a single open-end microstrip transmission line and a 1 × 8 PD, respectively, were used. The proposed antenna design was fabricated on a 120 × 60 mm